Materials and methods to enhance hematopoietic stem cells engraftment procedures

ABSTRACT

This disclosure is directed to the methods of enhancing hematopoietic stem cells (HSPC) and progenitor cell (HSPC) engraftment procedure. Treatment in vivo of a HSPC donor with compounds that reduce PGE 2  biosynthesis or PGE 2  receptor antagonists alone, or in combination with other hematopoietic mobilization agents such as AMD3100 and G-CSF, increases the circulation of available HSPCs. Compounds that reduce the cellular synthesis of PGE 2  include non-steroidal anti-inflammatory compounds such as indomethacin. Treatment ex vivo of HSPC with an effective amount of PGE 2  or at least one of its derivatives such as 16,16-dimethyl prostaglandin E 2  (dmPGE 2 ), promotes HSPC engraftment. Similar methods may also be used to increase viral-mediated gene transduction efficacy into HSPC.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 14/638,676 filed on Mar. 4, 2015, which was acontinuation application of U.S. patent application Ser. No. 13/128,074filed on Jul. 18, 2011, which was the which is the National Stage ofInternational Application No. PCT/US2009/063654 filed on Nov. 6, 2009,which claims the benefit of U.S. Provisional Patent Application No.61/112,018 filed on Nov. 6, 2008, the disclosures of which are expresslyincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENTAL RIGHTS

This invention was made with government support under grant numbersHL069669, HL079654, and DK007519 awarded by National Institutes ofHealth. The U.S. government has certain rights in the invention.

FIELD OF INVENTION

Aspects and embodiment disclosed herein are related to materials andmethods for enhancing the engraftment of hematopoietic stem andprogenitor cells.

BACKGROUND

Hematopoietic stem and progenitor cell (HSPC) transplantation is aproven therapy for the treatment of certain malignant and nonmalignanthematologic diseases and metabolic disorders. Sources of HSPC fortransplantation include bone marrow, mobilized peripheral blood, andumbilical cord blood (UCB) (Goldman and Horowitz, 2002; Fruehauf andSeggawiss, 2003: Broxmeyer, et al., 2006). Physicians routinely performtransplants of bone marrow, mobilized peripheral blood stem cells andumbilical cord blood. These procedures require that sufficient numbersof hematopoietic stem and progenitor cells be harvested from healthynormal donors, or from patients before they develop a given condition orwhile they are in remission. The harvested materials are subsequentlyadministered to patients whose hematopoietic system and presumably itsdiseased or malformed tissues and cells have been eradicated. Aftertransplantation, the transplanted stem cells travel to or “home” to theappropriate bone marrow microenvironment niches, lodge within suchniches, proliferate and produce new stem cells, a process calledself-renewal (Porecha, et al., 2006; Broxmeyer, 2006; Hall et al.,2006). The cells also differentiate into lineage restricted progenitorcells and mature cells, thus restoring the blood forming hematopoieticsystem necessary for the health of the recipient. Progenitor cells areusually present in the transplanted materials and may be required inthese grafts in order to produce mature cells. However, since progenitorcells are not stem cells and cannot self-renew, they participate intransplant therapy for only a limited period of time.

Because the transplant procedure stresses the transplanted material, asuccessful transplant requires that sufficient cells be transplanted toaccount for cells killed or damaged during the procedure. This presentsa large problem for the transplant of umbilical cord blood grafts asthese grafts include very limited numbers of stem cells. For thisreason, cord blood grafts usually cannot be used to successfullytransplant adults. Similarly 10-25% of patients and normal donors failto mobilize sufficient cells for use in transplant procedures. In somepatient populations, particularly those treated with somechemotherapeutic agents, failure to mobilize is seen in upward of 50% ofpatients. In general, the more cells that can be transplanted thegreater the likelihood that the transplant will be successful, forexample, current best practices recommend that peripheral blood stemcell transplantation procedures typically require minimum administrationof approximately 2 million CD34⁺ cells per kilogram of recipient patientbody weight, the more CD34+ cells that can be acquired and subsequentlytransplanted, the better the patient outcome (Pulsipher, 2009).

Inadequate stem cell number, inability to migrate/home to appropriatemarrow niches, or poor engrafting efficiency and self-renewal ofhematopoietic stem and progenitor cells can adversely affect transplantoutcome, measured by the multi-step process of repopulation. Numerousapproaches have been tried to try and expand the number of humanhematopoietic stem and progenitor cells within isolated grafts in exvivo settings with limited success. Strategies to improve HSPCtransplantation efficacy is needed to overcome the challenge faced bythe medical profession. Some aspects and embodiments of the inventiondisclosed herein address this need.

SUMMARY

Some aspects of the disclosure are directed to the enhancement ofhematopoietic stem and progenitor cells harvesting and/or engraftment,some of these aspects include, but are not limited to, ex vivo survival,self-renewal and homing to appropriate marrow niches to increase thesuccess rate for hematopoietic stem and progenitor cell therapy.

Some aspects of the disclosure include methods directed towardsincreasing the number of hematopoietic stem and progenitor cells withlong-term repopulation capabilities harvested from a donor. Some ofthese methods comprise the steps of: identifying a compound thatinhibits the biosynthesis of a prostaglandin, such as prostaglandin E,or a compound that antagonizes at least one prostaglandin receptorinvolved in the prostaglandin response; and providing a pharmaceuticallyeffective amount of the compound(s) to the donor prior to harvestinghematopoietic stem and progenitor cells from the donor's peripheralblood or bone marrow. In one embodiment, the application ofprostaglandin E biosynthesis inhibitor and/or prostaglandin E's receptorantagonist is coupled with one or more clinically approved hematopoieticstem and progenitor cell mobilization agents, for example,Granulocyte-Colony Stimulating Factor (G-CSF) , to increase the numberof hematopoietic stem and progenitor cells that can be collected byapheresis for hematopoietic graft transplantation. In one embodiment,the compound is selected from cylooxygenase inhibitors, including forexample, indomethacin(2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl}aceticacid) or a pharmaceutically acceptable salt thereof. In still otherembodiments the cyclooxygnease inhibitor is chosen from the groupconsisting of, aspirin, ibuprofen, Celecoxib, Rofecoxib, Meloxicam,Etoricoxib, Valdecoxib, Naproxen, Diclofenac, Licofelone, Etodolac,Ketorolac or pharmaceutically acceptable salts thereof. In someembodiments the cyclooxygenase inhibitor acts on both COX-1 and COX-2often times with a preference for COX-2. Still another compound that canbe used in some embodiments is Meloxicam.

Other aspects of the disclosure include methods for enhancing aharvested hematopoietic stem and/or progenitor cells graft's long termrepopulation capability in a recipient. This may be of particularutility in situations in which the recipient has a compromisedhematopoietic system. The method comprises the steps of: a) harvesting agraft from a donor, wherein the donor has been treated with an effectiveamount of compound to inhibit the biosynthesis of prostaglandin E₂and/or antagonist of prostaglandin E₂ receptor; b) contacting the graftwith an effective amount of prostaglandin E₂ or one or more of itsderivatives ex vivo; and c) applying the treated graft to the recipient.In one embodiment, the method further comprises the step of supplying aneffective amount of prostaglandin E₂ or one of its derivatives or anymolecule that has PGE₂ activity to a transplant recipient in order toenhance the homing of the graft materials to their appropriatetherapeutic niches.

Other aspects of the disclosure include methods for enhancing viraitransduction efficacy in stem cells. Some of these methods may includethe steps of: providing a viral vector that contains at least one geneof interest for transduction; providing at least one stem cell that hasbeen ex vivo treated by an effective amount of prostaglandin E₂ or itsderivatives, and transfecting the viral vector to the PGE₂ or itsderivative treated stem cell.

Some embodiments include methods of enhancing the mobilization ofhematopoietic stem and/or progenitor cells, comprising the steps of:identifying a source of hematopoietic stem and/or progenitor cells;providing a compound that reduces the biosynthesis and/or activity ofPGE₂; and contacting the source of hematopoietic stem and/or progenitorcells with an effective amount of said compound that reduces the cellsPGE₂ biosynthesis and/or activity. In some embodiments the compound thatreduces PGE₂ activity is a non-steroidal antiinflammatory compound,wherein the non-steroidal anti-inflammatory compound acts on bothcyclooxygenase-1 and cyclooxygenase-2. In some embodiments thenonsteroidal anti-inflammatory compound acts primarily oncyclooxygenase-2. In some embodiments the non-steroidalanti-inflammatory compound is selected from the group consisting of:aspirin, celecoxib, rofecoxib, etoricoxib, valdecoxib, ibuprofen,naproxen, diclofenac, etodolac, ketrolac and licofelone. In still otherembodiments the non-steroidal anti-inflammatory compound is indomethacinand in yet other embodiments the non-steroidal anti-inflammatorycompound is meloxicam.

In some embodiments the non-steroidal anti-inflammatory compound isadministered to a patient for a period of time overlapping withco-treatment with at least one additional compound that enhances themobilization of hematopoietic stem and progenitor cells. In someembodiments the compound that enhances the consisting of: G-CSF andplerixafor. In some embodiments the non-steroidal anti-inflammatorycompound is administered to a patient for at least 3 days.

Still other embodiments include methods of enhancing the mobilization ofhematopoietic stem and/or progenitor cells from a donor, comprising thesteps of: providing a compound that is an antagonist of at least onePGE₂ receptor; and administering an effective amount of said compound toa hematopoietic stem or progenitor cell donor prior to harvestinghematopoietic stem or progenitor cells from the donor. In someembodiments the antagonist of at least one PGE₂ receptor is selectedfrom the groups consisting of:

N-[[4′-[[3-butyl-1,5-dihydro-5-oxo-1-[2-(trifluoromethyl)phenyl]-4H-1,2)4-triazol-4-yl]methyl][1,r-biphenyl]-2-yl]sulfonyl]-3-methyl-2-thiophenecarboxamide(L-161, 982) and4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[fJisoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide(GW627368X).

Yet other embodiments include engrafting hematopoietic stem and orprogenitor cells into recipient, comprising the steps of: harvesting agroup of cells that includes hematopoietic stem and progenitor cellsfrom a source that has been treated with at least one compound thatreduces PGE₂ biosynthesis and/or activity in the source; contacting theset hematopoietic stem cells with a compound with PGE₂ activity ex vivo;and transplanting said hematopoietic stem and progenitor cells contactedwith said compound that increases PGE₂ activity ex vivo, into arecipient. In some embodiments the hematopoietic stem cells are drawnfrom a bone marrow donor. While in still other embodiments thehematopoietic stem cells are harvested from a sample of blood drawn froma blood donor. And still other embodiment the cells are drawn from anumbilical cord or a placenta.

Some embodiments include method of increasing hematopoietic stem and/orprogenitor cell engraftment rates, comprising the steps of providing acompound with PGE₂ activity; and contacting the compound with PEG₂activity with a population of hematopoietic stem and/or progenitor cellsex vivo. In some embodiments the compound with PGE₂ activity, isselected from the group consisting of any E series prostaglandin or anyderivative of an E series prostaglandin, such as PGE₁, PGE₂, PGE₃ or thedimethyl derivatives of PGE₁, PGE₂, PGE₃, including, for example,dimethyl 16, 16-dimethyl PGE₂. In some embodiments the compound havingPGE₂ activity is contacted with the hematopoietic stem and/or progenitorcell population for at least 1 hr. Some embodiments include the steps ofwashing the hematopoietic stem and/or progenitor cells that were incontact with the compound having PGE₂ activity, at least once with abuffer that is substantially free of PGE₂ activity. While still otherembodiments further include the step of: introducing the hematopoieticstem and/or progenitor cells that were in contact with the compoundhaving PGE₂ activity into a patient.

These and other features, aspects and advantages of the presentinvention may be better understood with reference to the following nonlimiting drawings, description and claims.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A. Outline of an experiment to test the effect of PGE2 enhanceshematopoietic stem cell engraftment (upper panel); a representative flowplot illustrating the populations of CD45.1 and CD45.2 cells (lowerpanel).

FIG. 1B. Graph of percent negative cells versus number of transplantedcells (upper panel); Scatter graph of percent chimerism versuscompetitor ratio (lower conditions (lower right panel).

FIG. 1C. Table summarizing Repopulating Cell Frequency plotted over 20weeks for cells treated with and without dmPGE2.

FIG. ID. Representative FACS plots of multi-lineage reconstitution(myeloid, B and T-lymphoid, upper left panel). Plot of counts per CD3(upper row right panel). Middle row, bar graphs percent of Total WBCmeasured at 32 weeks in primary recipients (left panel) and 12 weeks insecondary recipients (right panel).

Plot of percent chimerism measured at 20 weeks in primary recipients and12 weeks in secondary recipients (bottom panel).

FIG. 2A. Representative FACS gating of MACS microbead depleted Lin^(neg)marrow showing c-kit⁺ and Sca-1⁺ gating of Lin^(neg) gated cells (leftside panel). Count plotted for different EP receptors (middle panels)and change in mRNA versus cycle number potted for different receptors(right panels).

FIG. 2B. Representative FACS SSC versus CD34 (left panel); Countsplotted for different EP receptors (middle panel) and Change in mRNAplotted versus Cycle Number for different receptors (right panel).

FIG. 3A. Outline of experiment (top); % CFSE+ plotted for differenttreatments (bottom panel).

FIG. 3B. Diagram illustrating experimental protocol; percent homingefficiency (lower left panel) and Fold Change (lower right panel)measured after exposure to 16,16-dimethyl prostaglandin E₂ (dmPGE₂) andvarious controls.

FIG. 3C. Outlines of experiments (left panels); FACS plots of CD45.2versus CD45.1 for different treatments (middle panels); percent homingefficiency plotted for different treatments (right panel).

FIG. 4A. representative flow plot of CXCR4 receptor expression isotypecontrol shown in gray (top row); Bar graph illustrating results of pulseexposure of murine and human HSPC to PGE₂ on CXCR4 expression, change inCXCR4 plotted for different conditions (bottom row).

FIG. 4B. Bar graph of percent homing efficiency plotted for differenttreatments.

FIG. 5A. Percent of Annexin V+ SKL plotted as a function of dmPGE₂concentration.

FIG. 5B. Fold increase in Survivin plotted for different conditions.

FIG. 5C. Percent change normalized to control activity plotted fordifferent times of exposure to dm PGE₂.

FIG. 6A. Representative flow plots showing DNA content (7AAD staining)of gated SKL cells, the percent of SKL in S+G2M phase, and the foldincrease in cycling of dmPGEΣ-treated SKL (left panel); the chart showscombined data from 3 experiments (right panel).

FIG. 6B. Outline of experiment and bar graph plotting of fold increasein homed SKL cells in S+G₂/M measured with different treatments. Cartoonshowing experimental protocol (left panel); bar graph Fold Increase inS+G₂/M measured with and without dmPGE₂.

FIG. 7A. Bar graph plotting CFU-GM per mL of blood measured afterdifferent treatments.

FIG. 7B. Bar graph plotting CFU-GM per mL of blood measured afterdifferent treatments.

FIG. 8. Table summarizing data illustrating that PGE₂ effects thecycling of SLAM SKL.

FIG. 9. Graph of migration control and dmPGE₂-treated cells versus SDF-1concentration.

FIG. 10. Graph of percent CD34+ cell migration versus SDF-1concentration and/or AMD3100(1,1′-[1,4-Phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane]octohydrobromidedihydrate) marketed under the trade name MOZOBIL®.

FIG. 11. Bar graph of percent homing efficiency of SKL cells versustreatment with dmPGE₂ and/or AMD3100 and various controls.

FIG. 12. FACS plot (left panel); and bar graph (right panel)illustrating Fold change in SKL cycling measured with and withoutdmPGE₂.

FIG. 13. Outline of a an experimental protocol (left panel); a bar graphshowing an increase in S+G2/M measured with and without added dmPGE₂.

FIG. 14. Graph of percent chimersim measured in serial transplants overtime after initial exposure to dmPGE₂ (squares) or control (vehicle,diamonds).

FIG. 15. Bar graph CFU-GM per ml of blood measured with vehicle (lightgray) indomethacin (dark gray) or baicalein (gray) left panel; graph ofdata collected with G-CSF (light gray); G-CSF plus indomethacin (grayhatch) or G-CSF plus baicalein (gray).

FIG. 16. Bar graph Fold Increase in CFU per ml of Blood over G-CSFmeasured with G-CSF and G-CSF plus indomethacin.

FIG. 17. Bar graph of phenotypic analysis of mobilized cells either SKLcells (left side) or SLAM SKL cells (right side) measured aftertreatment with either G-CSF or G-CSF plus indomethacin.

FIG. 18. Bar graph CFU-GM per ml of Blood plotted with either vehicle orindomethacin (left panel); or AMD3100 or AMD3100 plus indomethacin(right panel).

FIG. 19. Bar graph CFU-GM per ml of Peripheral Blood plotted measuredafter treatment with vehicle, indomethacin, AMD3100; G-CSF; AMD3100plusGROBeta; AMD3100 plus indomethacin or G-CSF plus indomethacin.

FIG. 20. Bar graph CFU-GM per ml of Blood after treatment with vehicle(clear), G-CSF (black), G-CSF plus meloxicam((8E)-8-[hydroxy-[(5-methyl-1,3-thiazol-2-yl)amino]methylidene]-9-methyl-10,10-dioxo-10λ⁶-thia-9-azabicyclo[4.4.0]deca-1,3,5-trien-7-one)(light gray), or G-CSF plus indomethacin (gray).

FIG. 21. Bar graph Competitive Repopulating units measured with eitherG-CSF (light gray) or G-CSF plus indomethacin (gray) (left panel); andMFI CXCR4 on SKL cells measured with vehicle, G-CSF; no Stagger, 1 daystagger or 2 day stagger (right panel).

FIG. 22. Graph of percent Chimerism versus PBMC: BM ratio measured witheither G-CSF (diamonds) or G-CSF plus indomethacin (squares) (leftpanel); CRU scaled to 2 million PBMC measured with either G-CSF (lightgray) or G-CSF plus indomethacin (gray).

FIG. 23. Graph of PMN versus days after transplant of PBMC mobilized byG-CSF (diamonds) or G-CSF plus Metacam (meloxicam)(squares).

FIG. 24. Graph of PLT versus days after transplant of PBMC mobilized byG-CSF (diamonds) or G-CSF plus Metacam (meloxicam)(squares).

FIG. 25. Cartoon summarizing experiment designed to test the effect oftreating baboons with either G-CSF alone or G-CSF plus meloxicam.

FIG. 26. Plots of CD34+ Cells (left side) or CFU-GM (right side) per mLof blood drawn from 4 different baboons treated with either G-CSF orG-CSF plus meloxicam.

FIG. 27. Bar graph CFU-GM per mL of blood tested using differentcompounds that vary in their selectivity for either COX-1 or COX-2.

FIG. 28. Bar graph of Fold Changes in CFU-GM over G-CSF per mL of bloodtested after treating cells with G-CSF or G-CSF plus different amountsof either aspirin or ibuprofen.

FIG. 29. Bar graph CFU-GM per mL of blood measured in the peripheralblood after treatment with either G-CSF or different levels ofmeloxicam.

FIG. 30. Bar graph CFU-GM per Femur measured in bone marrow aftertreatment with either G-CSF or different levels of meloxicam.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended, such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates.

Prostaglandin E2 (PGE2) is an abundant physiological eicosanoid and aknown mediator of cancer, inflammation, and numerous other physiologicalsystems. The roles for PGE₂ in hematopoiesis have been explored byvarious research teams, but the outcomes are difficult to reconcile. Forexample, in vitro and in vivo studies demonstrate that PGE2 cannegatively regulate myelopoiesis: PGE₂ promotes BFU-E and CFU-GEMMcolony formation and enhances proliferation of CFU-S and CFU-GM. On theother hand, PGE₂ can stimulate HSPC and have biphasic effects onhematopoiesis: Short-term ex vivo PGE₂ treatment of marrow cells wasshown to stimulate the production of cycling human CFU-GM from apopulation of quiescent cells, possibly stem cells or more primitiveprogenitor cells. Further, recently, it was shown that ex vivo exposureto 16, 16-dimethyl PGE₂ increased the repopulating capacity of murinebone marrow cells and kidney marrow recovery in zebrafish (North et at,2007). These studies implicate PGE₂ in the regulation of hematopoiesis,but fail to link PGE₂ to hematopoietic stem cell homing. Rather, theprevious studies tend to indicate PGE₂ is involved in modulation of HSPCdifferentiation, and PGE₂ has no direct effect on cell homing.

As demonstrated herein PGE₂ has direct and stabilizing effects onlong-term repopulating HSPC and facilitates engraftment by enhancingsurvival, homing, and proliferation of self-renewing HSPC.

One aspect disclosed herein is the inhibition of cyclooxygenase activitywhich increases the frequency of hematopoietic stem and progenitor cellcirculation in the peripheral blood system. In one non-limiting example,administration of cyclooxygenase inhibitors, for example, 50 microgramsof indomethacin daily, by oral or systemic routes to hematopoieticdonors one day prior to and with each day they receive a dose ofmobilizing agent, enhanced the mobilization of stem cells and progenitorcells in the periphery. Concurrent use of cyclooxygenase inhibitor, forexample, Indomethacin, with clinically approved mobilizing agent, forexample, G-CSF, produces a synergistic effect to mobilize progenitorcells.

The mobilization of hematopoietic stem cell and progenitor cell can alsobe achieved by providing the donor with an effective amount of aprostaglandin E receptor antagonist.

Some aspects disclosed show that ex vivo exposure to PGE₂ enhances HSPCfrequency after transplantation and provides a competitive advantage forPGE₂-treated HSPC. Treating bone marrow stem cells with PGE₂ ex vivoenhances total stem cell engraftment in mice, resulting in enhanced stemcell survival, increased stem cell homing efficiency and increased stemcell self-renewal. Enhancement of HSPC frequency induced by dmPGE₂ wasdemonstrated by using a limiting dilution competitive transplantationmodel that compared engraftment of control and dmPGE₂-treated cells indirect head-to-head analysis within the same animal. For example,un-treated hematopoietic grafts or purified hematopoietic stem cellpopulations (e.g., SKL cells in mice or CD34+ cells in humans) wereincubated with authentic PGE₂ or the more stable analog 16, 16-dimethylPGE₂ (or any additional active PGE analogue) on ice at concentrations of0.001-10 microMolar PGE₂ per 1-10 million cells in 1 ml of culturemedium, e.g. IMDM, for 1-6 hrs. After incubation, the cells were washed3 times in sterile saline and administered to recipients, intravenously.This process demonstrated a ˜4-fold competitive advantage of PGE₂-pulsedHSPC based upon calculation of HSPC frequency by Poisson statistics andanalysis of competitive repopulating units (CRU). Frequency analysisdemonstrates equivalent reconstitution using one-fourth the number ofPGE₂ treated cells vs. control cells. In addition, full hematopoieticreconstitution was observed in secondary transplant recipients usingeither control or PGE₂-treated cells, indicating no adverse impact ofPGE₂ on HSPC self-renewal. In fact, a trend towards increased LTRCactivity was seen, indicating that the enhancing effect of short-termPGE₂ exposure on HSPC observed in primary transplants was long lasting,since no additional treatment was performed on cells or animals beforeserial-transplant. Enhanced engraftment of PGE2-treated cells was stableover 28 weeks. Analysis in secondary transplanted animals 90 dayspost-transplant demonstrated full multi-lineage reconstitution andcontinued higher HSPC frequency, indicating a stable effect ofshort-term PGE₂-treatment on long-term repopulating HSPC.

Enhanced engraftment can result from changes in HSPC frequency, homing,survival and/or proliferation. It was suggested by North, et.al. thatPGE₂ did not affect HSPC homing; however, their studies did notspecifically assess HSPC. Unexpectedly, as demonstrated herein thePGE2-induced enhanced HSPC frequency was stable throughout a >20 weekperiod and was maintained in secondary transplants. Direct comparison incompetitive transplant models showed that short-term exposure of HSPC toPGE₂ produced a ˜4-fold competitive advantage. Although totaltransplanted cells had no difference in homing efficiency betweencontrol and PGE2-treated cells, enhanced homing efficiency ofPGE₂-treated, sorted SKL cells was observed, strongly suggesting PGE₂has a direct effect on HSPC homing.

These results suggest PGE₂'s greater effect for HSPC or HSPC long termrepopulation ability, rather than only a short term effect as proposedby previous studies.

One possibility, offered by way of explanations and not limitation, isthat the effects of PGE₂ on HSPC function might be mediated throughupregulation of the alpha-chemokine receptor CXCR4 chemokine receptor,implicated in HSPC homing and self-renewal, and the inhibitor ofapoptosis protein Survivin, which regulates HSPC survival andproliferation.

Flow cytometry and QRT-PCR shows expression of all 4 PGE₂ receptors(EP1-EP4) on Sca-1⁺, c-kit⁺, Lineage^(neg) (SKL) murine marrow cells andon CD34⁺ human cord blood cells (UCB) with no overt differences inreceptor subtype expression. When analyzing several functionalproperties relevant to HSPC function, a significant increase in CXCR4expression on both SKL (26.8%) and CD34⁺ UCB (17.3%) was seen after PGE₂exposure, with significant upregulation of CXCR4 mRNA at −6 hourspost-exposure. Increased CXCR4 was coincident with an ˜2-fold increasein in vivo marrow homing efficiency of PGE₂-treated grafts and wasobserved with un-manipulated bone marrow (p<0.001, 3 expts, n=6mice/group/expt, assayed individually) and with purified SKL cells inhead-to-head competition in the same animal (p<0.001, 2 expts, n=5mice/group/expt, assayed individually), indicating a direct effect OfPGE₂ on HSPC. The increase in homing efficiency was significantlyreduced by treatment with the selective CXCR4 antagonist AMD3100.

PGE₂ treatment increased SKL cell CXCR4 mRNA and surface expression. Inaddition, the CXCR4 antagonist AMD3100 significantly reduced theenhancing effect of PGE₂ on homing, suggesting that enhanced CXCR4expression and chemo-attraction to marrow SDF-I is largely responsiblefor enhanced homing, although additional effects on adhesion moleculeexpression or function cannot be excluded.

One aspect disclosed herein, is that PGE₂ treatment of a recipientenhances survival of stem cells transplanted into recipients in vivo.Parenteral administration of PGE₂ or active analogs to recipients at thetime of transplant and to continue daily administration to enhance stemcell might increase the survival of transplanted HSPC. For example, PGE₂or its active analogue could be administered as 0.0001-10 micro Molar topatients immediately prior to and daily after receiving a hematopoieticgraft.

PGE₂ treatment in vitro results in an increase in the proportion of SKLcells actively in cell cycle within 24 hours post-treatment. Inaddition, transplantation of PGE₂-treated cells in BrdU treatedrecipient mice showed ˜2-fold more donor SKL cells in S+G₂/M phase ofthe cell cycle compared to transplanted cells pulsed with vehicle only.

Survivin is thought to be required for HSPC to enter and progressthrough cell cycle and Survivin's deletion in conditional knockout miceindicates it is required for HSPC maintenance. Studies reported hereinfound elevated mRNA and protein levels of Survivin, with concomitantreduced active caspase-3, a protease that mediates apoptosis, inPGE₂-treated SKL cells. Survival assays indicated that PGE₂dose-dependently decreased apoptosis of SKL cells in vitro, coincidentwith a 1.7 fold increase in Survivin protein expression and a decreasein active caspase-3 (23-59% decrease; 24-72 hours post exposure).

It is likely that enhanced HSPC survival, mediated through Survivin,contributes to enhanced engraftment. Pulse exposure to PGE₂ increasesthe proportion of HSPC in cell cycle by ˜2-fold, with increasedfrequency of HSPC, CRU and homing of BrdU⁺ SKL cells and maintenance ofenhanced HSPC frequency in primary and secondary transplants. Onenon-limiting explanation of these results is that PGE₂ pulse-exposuremay initiate a single round of HSPC self-renewal. For example, EP2 andEP4 receptor activation is associated with phosphorylation of glycogensynthase kinase-3 (GSK-3) and increased β-catenin signalling (Hull etal., 2004; Regan, 2003), which is downstream of the Wnt pathway, whichhas been implicated in HSPC survival and self-renewal (Fleming et al,2008; Khan and Bendall, 2006). Signalling by PGE₂ possibly through EP4but not limited exclusively to EP4 might directly increase β-catenin.Synergistic cross-talk between COX-2 and Wnt pathways has been suggested(Wang et al, 2004).

Survivin also facilitates HSPC cell cycling through _(p)21^(WAF1/CDKN1)(Fukuda et al, 2004), known to be involved in HSPC function (Cheng etal, 2000), and blocks caspase-3 activity (Li et al, 1998; Tamm et al,1998). Recently, p21 was implicated in HSPC self-renewal (Janzen et al,2008). One finding drawn from the studies reported herein is that PGE2up-regulates Survivin and decreases caspase-3 suggesting that theSurvivin pathway may be involved in the effects of PGE₂ on increasedself-renewal. It is also interesting to note that Survivin (Peng et al.,2006) and CXCR4 (Staller et al, 2003; Zagzag et al, 2005) transcriptionare up-regulated by the transcription factor hypoxia-inducible factor-1alpha (HIF-1 alpha), which can be stabilized by PGE₂ (Liu et al, 2002;Piccoli et al, 2007), possibly linking some PGE₂ responsive pathwayswith cell survival, homing, and proliferation/self-renewal of HSPC.

These studies suggest that the ˜4-fold increase in HSPC frequencyobserved after PGE₂ treatment results from a ˜2-fold or more homing ofHSPC to recipient marrow with a ˜2-fold more HSPC undergoingself-renewal. These results may help to define novel mechanisms ofaction whereby PGE₂ enhances HSPC function and they suggest unexpectedtherapeutic approach for facilitating hematopoietic transplantation,particularly for hematopoietic grafts in which a limiting number ofcells results in a poor potential for engraftment.

One aspect disclosed herein is a method for enhancing the viraltransduction efficacy in stem cell gene therapy. The ex vivo PGE₂treatment of stem cells increased the self-renewal division and survivalof such cells, which is an important factor for successful viral vectormediated gene integration. PGE₂ promoted stem cell self-renewaldivision/survival can be incorporated in current stem cell transductionprotocols, thus increasing the overall gene transduction efficacy instem cell gene therapy.

Reported herein are some methods of using PGE₂ to enhance HSPCengraftment, a multistep process that includes the mobilization of donorcells, the maintenance of HSPCs and the homing of HSPC in the recipientbody. Under some conditions these methods result in a 4-fold increase inHSPC frequency and engraftment results possibly, for example, from thecumulative effect of a 2-fold increase in HSPC homing and a 2-foldincrease in HSPC cell cycle activity under the direct influence of PGE₂.Although the precise signaling pathways are yet to be determined, onenon-limiting explanation for this effect is that enhanced engraftment isdue to up-regulation of factors such as CXCR4 and Survivin.

The ability of PGE₂ to improve the homing and the survival and/orproliferation of HSPC may be clinically significant, especially insettings in which HSPC numbers are limiting, e.g. UCB and some mobilizedPB products, or for viral gene transduction in stem cell gene therapy.Our limiting dilution transplant studies illustrate that equivalentengraftment results can be achieved with one- fourth the number ofPGE2-treated cells compared to controls that are not so treated. Theseresults demonstrate the utility of using PGE₂ under conditions in whichHSPC numbers are limiting. While all four EP receptor subtypes appear tobe expressed on HSPC, it is not clear which of these receptors (or ifall of them) are involved in the engraftment function. It is consistentwith these results that enhanced engraftment/recovery can be achieved byadministering PGE₂ in vivo or if PGE₂ used in vivo can furtherfacilitate engraftment of HSPC exposed to PGE₂ ex vivo.

MATERIALS AND METHODS Materials

Mice C57B1/6 mice were purchased from Jackson Laboratories (Bar Harbor,Me., USA). B6.SJL-PtrcAPep3B/BoyJ (BOYJ) and F1 C57B1/6/BOYJ hybridswere bred in-house. All animals were housed in micro-isolator cages withcontinuous access to food and acidified water. Mice used in transplantstudies received Doxycycline feed at time of radiation and for 30 dayspost-transplant. The Animal Care and Use Committee of Indiana UniversitySchool of Medicine approved all animal protocols.

Flow Cytometry All antibodies were purchased from BD Biosciences unlessotherwise noted. For detection and sorting of murine KL and SKL cells,streptavidin conjugated with PE-Cy7 (to stain for biotinylated MACSlineage antibodies (Miltenyi Biotech, Auburn, Calif.)), c-kit-APC,Sca-1-PE or APC-Cy7, CD45. 1-PE and CD45.2-FITC were used. UCB CD34⁺cells were detected using anti-human-CD34-APC. For multilineageanalysis, APC-Cy7-Mac-1 , PE-Cy7-B-220 and APC-CD3 were used. EPreceptors were detected with anti EP1, EP2, EP3 and EP4 rabbit IgG(Cayman Chemicals) and secondary staining with FITC-goat-anti-rabbit IgG(Southern Biotech, Birmingham, Ala.). CXCR4 expression was analyzedusing streptavidin-PECy7, c-kit-APC, Sca-1-APC-Cy7, and CXCR4-PE.Apoptosis was measured with FITC-Annexin-V. For Survivin and activecaspase-3 detection, cells were permeabilized and fixed using theCytoFix/CytoPerm kit (BD) and stained with anti-active-caspase-3-FITCFlow Kit (BD) or Survivin-PE (R&D Systems).

For cell cycle analysis, cells were stained with 7AAD or the FITC-BrdUFlow Kit (BD). All analyses were performed on a LSRII and sorting wasperformed either a FACSAria or FACSVantage sorter (BD). Cell Quest Proand Diva software (BD) were used for data acquisition and analysis.

Methods

Limiting Dilution Competitive and Non-Competitive Transplantation

WBM cells (CD45.2) were treated on ice for 2 hours with either 1microMolar dmPGE₂ (Cayman Chemical, Ann Arbor, Mich.) per 1×10⁶ cells or0.01% ETOH in sterile, non-pyrogenic PBS. After incubation, cells werewashed twice and mixed with 2×10⁵ congenic CD45.1 competitor marrowcells at ratios of 0.075:1, 0.25:1, 1 :1, and 2.5:1 and transplantedinto lethally irradiated CD45.1 mice (1100-cGy split dose) by tail-veininjection (5 mice per dilution). CD45.1 and CD45.2 cells in PB weredetermined monthly by flow cytometry. For head-to-head competitivetransplants, WBM from CD45.1 mice and CD45.2 mice were treated withvehicle or dmPGE₂ and mixed with 2×10⁵ competitor marrow cells fromCD45.1/CD45.2 mice at ratios of 0.075:1, 0.25:1, 1:1, and 2.5:1 andtransplanted into lethally irradiated CD45.1/CD45.2 mice. The proportionof CD45.1, CD45.2, and CD45.1/CD45.2 cells in PB was determined monthly.HSPC frequency was quantitated by Poisson statistics using L-CALCsoftware (Stem Cell Technologies, Vancouver BC, Canada) with <5%contribution to chimerism considered a negative recipient. Competitiverepopulating units (CRU) were calculated as described (Harrison, 1980).For secondary transplants, 2×10⁶ WBM from previously transplanted F1Hybrid mice at the 1:1 ratio at 20 weeks post-transplant were injectedinto lethally irradiated F1 Hybrid mice in non-competitive fashion andPB chimerism and tri-lineage reconstitution evaluated monthly.

Analysis of HSPC homing to bone marrow in vivo CD45.2 WBM was labeledwith CFSE (Molecular Probes, Eugene, Ore.) washed and treated on icewith either 1 microMolar dmPGE₂ or vehicle. After treatment, cells werewashed and 2×10⁷ cells transplanted into lethally irradiated CD45.2mice. After 16 hours, femurs and tibias were flushed, and a proportionof mouse marrow Lin^(t) cells depleted using MACS microbeads (MiltenyiBiotech), stained with fluorochrome-conjugated-antibodies specific forbiotin (lineage), c-kit (K), and Sca-1 (S) and the total number Of CFSE⁺WBM (non lineage depleted), KL and SKL cells determined. For congenichoming studies, Lin^(neg) CD45.1 cells were treated on ice with 1microMolar dmPGE2, vehicle, or PBS. After incubation, cells were washedand 2×10⁶ cells transplanted into CD45.2 mice. After 16 hours, recipientbone marrow was harvested, lineage depleted, stained, and donor CD45.1SKL cells determined. For competitive, head-to-head homing studies usingsorted SKL cells, Lin^(neg) cells from CD45.2 and CD45.1 mice were FACSsorted, cells treated with either dmPGE2 or vehicle for 2 hours, washedand 3×10⁴ CD45.1 (vehicle or dmPGE2 treated) plus 3×10⁴ CD45.2 (dmPGE₂or vehicle treated) SKL cells transplanted into lethally irradiated F1Hybrid mice. To evaluate the role of CXCR4 in homing studies, Lin^(neg)CD45.2 cells were treated on ice with vehicle or 1 microMolar dmPGE2plus 10 microMolar AMD3100 (AnorMed Inc., Vancouver, BC, Canada) and2×10⁶ treated cells injected into lethally irradiated CD45.1 mice. HomedSKL cells were analyzed 16 hours post-transplant.

Expression of EP receptors, CXCR4 and Survivin Replicate Lin^(neg) cellsamples from CD45.2 mice were stained for SKL and each of the EPreceptors and surface receptor expression on KL and SKL cells determinedby FACS. For human EP receptors, UCB was obtained from Wishard Hospital,Indianapolis, Ind. with Institutional Review Board approval. Mononuclearcells were isolated on Ficoll-Paque™ Plus (Amersham Biosciences) andCD34⁺ cells positively selected with MACS microbeads (Miltenyi Biotech)(Fukuda and Pelus, 2001). Replicate cells were stained for CD34 and eachof EP receptors and surface expression determined by FACS. To evaluateCXCR4, Survivin and active caspase-3, Lin^(neg) cells or CD34⁺ UCB weretreated on ice with either 1 micrMolar dmPGE2 or vehicle control for 2hours, washed, and then cultured in RPMI-1640+ 10% FBS at 37° C. for 24hours. Cells were stained for SKL (murine cells) and CXCR4, Survivin,and/or active caspase-3, as described above, and analyzed by FACS.

Cell Cycle Analysis For in vitro cell cycle analysis, Lin^(neg) cellswere treated with either 1 microMolar dmPGE₂ or vehicle for 2 hours,washed, and cultured in Stem Cell Pro Media (Stem Cell Technologies)with rmSCF (50 ng/ml) (R&D Systems, Minneapolis, Minn.), rhFlt-3 andrhTPO (100 ng/ml each) (Immunex, Seattle, Wash.). After 20 hours, cellswere stained for SKL, fixed and permeabilized, and stained with 7AAD (BDBiosciences, San Jose, Calif.). The proportion of SKL cells in S+G2/Mphase was determined by measuring DNA content by FACS. For in vivo cellcycle analysis, CD45.2 mice were lethally irradiated and transplantedwith 5×10⁶ Lin^(neg) cells from CD45.1 mice treated with either 1microMolar dmPGE₂ or vehicle for 2 hours. At the time of transplant,recipient mice received 1 milligram/mL BrdU (Sigma Aldrich, St. Louis,Mo.) in drinking water and 1 mg per mouse BrdU LP. After 16 hours,recipient marrow was isolated, lineage depleted, and stained for CD45.1,SKL and BrdU. The proportion of homed (CD45.1⁺) SKL cells that wereBrdU⁺ was determined by FACS in individual mice.

Apoptosis Assay Lin^(neg) cells were treated on ice with 0.1 nanoMolarto 1 microMolar dmPGE2 or vehicle control, washed and incubated inRPMI-1640+2% FBS, without growth factors at 37° C. to induce apoptosis.After 24 hours, cells were stained for SKL and Annexin-V and theproportion of Annexin-V⁺ SKL cells was determined by FACS.

Reverse Transcription and QRT-PCR Total RNA was obtained using theabsolutely RNA purification kit (Stratagene, La Jolla, Calif.). Aconstant amount of RNA was reverse transcribed with random primers(Promega, Madison, Wis.) and MMLV-reverse transcriptase (Promega) in avolume of 50 micro Liter with 1 milliMolar dNTPs and RNase inhibitor asdescribed (Fukuda and Pelus, 2001). DNase and RNase free water (Ambion,Austin, TX) was added to obtain a final concentration equivalent of 10nanogram RNA/microLiter and 5 microLiter used for QRT-PCR. Primers forSYBR Green QRTPCR were designed to produce an amplicon size of 75-150bp. QRT-PCR was performed in a total volume of 30 microLiter usingPlatinum SYBR Green qPCR supermix UDG with Rox (Invitrogen, Carlsbad,Calif.) in an ABI-7000 (Applied Biosystems, Carlsbad, CA), with anactivation step of 50° C. for 2 min, denaturation at 95° C. for 2 minand amplification for 45 cycles at 95° C.-15 sec, 50° C.-30 sec, 72°C.-30 sec, followed by dissociation to confirm that only one product wasobtained.

Nonsteroidal anti-inflammatory compounds that can be used to practicesome aspects of the invention, include, but are not limited to,compounds such as: Celecoxib (4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide) sold under the trade name Celebrex®;Rofecoxib (4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one) soldunder the trade name Vioxx®; Aspirin (2-acetoxybenzoic acid); Etoricoxib(5-chloro-6′-methyl-3-[4-(methylsulfonyl)phenyl]-2,3′-bipyridine);Valdecoxib (4-(5-methyl-3-phenylisoxazol-4-yl) benzenesulfonamide) soldunder the trade name BEXTRA®; Ibuprofen ((&S)-2-(4-isobutylphenyl)propanoic acid); Naproxen ((+)-(S)-2-(6-methoxynaphthalen-2-yl)propanoic acid); Diclofenac (2-(2-(2,6-dichlorophenylamino)phenyl)aceticacid) marketed under the trade name VOLTAREN®; Licofelone([6-(4-chlorophenyl)-2,2-dimethoyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl]aceticacid); Indomethacin(2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl}aceticacid) meloxicam((8E)-8-[hydroxy-[(5-methyl-1,3-thiazol-2-yl)amino]methylidene]-9-methyl-10,10-dioxo-10λ⁶-thia-9-azabicyclo[4.4.0]deca-1,3,5-trien-7-one)sold under the trade name Metacam; Etodolac(2-(1,8-Diethyl-4,9-dihydro-3H-pyrano[3,4-b]indol-1-yl)acetic acid);ketorolac ((±)-5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid,2-amino-2-(hydroxymethyl)- 1,3 -propanediol) marketed under the tradename Toradol.

Compounds that act as antagonists to at least one PGE₂ receptor include,but are not limited to, compounds available from Cayman Chemical Company(Ann Arbor, Mich., U.S.A.) or other lists maintained /sold by chemicalsupply companies.

Statistical Analysis All pooled values are expressed as Mean±SEM.Statistical differences were determined using the paired or unpairedtwo-tailed t-test function in Microsoft Excel (Microsoft Corp, Seattle,Wash.) as appropriate. As used herein, especially in some of thefigures, the terms, ‘dmPGE₂’ and ‘dmPGE’ are used interchangeably.

EXAMPLES 1. PGE₂ Increases Long-Term Repopulating HSPC Frequency andEngraftment

Using a limiting dilution competitive transplant model utilizing CD45.2and CD45.1 congenic grafts transplanted into CD45.1/CD45.2 hybrid mice,demonstrated that short-term exposure of HSPC to PGE2 produces long-termenhancement of HSPC and competitive repopulating unit (CRU) frequency.Referring now to FIG. 1A, bone marrow from CD45.1 or CD45.2 mice weretreated with vehicle or dmPGE2 respectively. CD45.1/CD45.2 hybrid marrowcells were used as competitors. Limiting dilutions were transplantedinto lethally irradiated (1100 cGys, split dose) CD45.1/CD45.2 hybridmice and chimerism in PB analyzed for 20 weeks. A representative flowplot detecting each cell population is shown (bottom panel).

Referring now to FIG. 1B, frequency analysis (top) for vehicle (red) ordmPGE₂ (blue) pulsed cells, determined by Poisson statistics, at 12weeks; Po=85,560 (vehicle) and Po=23,911 (dmPGE₂ treated). Chimerism inPB and CRU analysis is shown at 12 weeks (Mean ±SEM). Data represent 2pooled experiments, n=5 mice/group/expt, each assayed individually.

Referring now to FIG. 1C, HSPC frequency analysis in recipients ofvehicle or PGE2-treated bone marrow over 20 weeks. Fold change indicatesincrease in frequency of engraftment of dmPGE2-pulsed cells compared tovehicle.

Referring now to FIG. 1D, representative FACS plots of multi-lineagereconstitution (myeloid, B and T-lymphoid). Referring now to FIG. 1D,middle panels multi-lineage analysis for primary transplant (32 weeksleft panel) and a cohort of 4 mice that received transplants fromprimary transplanted mice at 20 weeks, with analysis 12 weeks later(right panel). Increased chimerism of dmPGE₂ treated cells vs. vehicleis shown for primary transplant at 20 weeks (time of secondarytransplant) and secondary transplant 12 weeks later (bottom panel). Datafor 20 week primary transplant were from 2 pooled experiments, n=5mice/group/expt, each assayed individually. Data for 12 week secondarytransplant, n=4 mice/group, each assayed individually.

Still referring to FIG. 1D, serial transplantation assesses self-renewaland expansion of HSPC in transplanted hematopoietic grafts. Toinvestigate the expansion of long-term repopulating cells (LTRC) exposedto dmPGE₂ and vehicle ex vivo, marrow was harvested from primarytransplanted animals at 20 weeks post- transplant and transplanted intosecondary recipients. Analyziz of PB 12 weeks after secondary transplantshowed multilineage reconstitution by cells from all transplanted mice,indicative of self-renewal of primary transplanted LTRC. The increase inchimerism resulting from dmPGE₂ exposure seen in primary donors was alsoseen in secondary transplants without any additional treatments. Inaddition, a trend towards increased competitiveness of HSPC previouslytreated with dmPGE₂ was observed in secondary transplants, with a slightbias towards myeloid lineage reconstitution.

This model permits quantitative comparison of engraftment andcompetitiveness of HSPC from control and dmPGE₂ treatment groups withinthe same animal (FIG. 1A), as well as endogenous repopulation of hostcells. At 12 weeks post-transplant, analysis of peripheral blood (PB)showed increased chimerism of dmPGE2-treated cells compared to vehicletreated cells, with ˜4-fold increase in HSPC frequency and competitiverepopulating units (CRU), recognized measures of long-term-repopulatingcapacity (FIG. 1B). Throughout 20 weeks of follow up post-transplant, an˜4-fold increase in HSPC frequency was maintained, indicating that theeffect of dmPGE₂ pulse exposure was stable (FIG. 1C). At 32 weekspost-transplant, reconstitution was seen for B- and T-lymphoid andmyeloid lineages in PB, with no discernible differences betweenuntreated competitor cells, dmPGE₂ or vehicle treated cells (FIG. 1D).

2. Murine and Human Hematopoietic Stem and Progenitor Cells (HSPC)Express PGE2 Receptors.

Reportedly, PGE₂ interacts with 4 specific, highly conserved G-proteincoupled receptors; EP1-EP4 (Sugimoto and Narumiya, 2007; Tsuboi et al,2002). EP receptor repertoire accounts for multiple, sometimes opposingresponses attributed to PGE₂ (Breyer et al, 2001). PGE2 receptor subtypeexpression on HSPC is not known previously. Referring now to FIG. 2A,analysis of EP receptors on c-kit⁺ Lin^(neg) (KL) cells, enriched forhematopoietic progenitor cells (HPC), and Sca-1⁺ c-kit⁺ Lin^(neg) (SKL)cells, enriched for HSPC, showed that all four EP receptors (EP3+, EP2,EP1 and EP4) were expressed. Referring now to FIG. 2A, (right panel) inaddition, QRT-PCR detected mRNA for all four EP receptors in FACS sortedKL and SKL cells. Referring now to FIG. 2A, (middle panel) dissociationcurves for EP3 showed several peaks, consistent with the known multiplesplice variants of EP3 (Namba et at, 1993). No significant quantitativedifferences in surface expression or mRNA levels between any of the EPreceptor subtypes was seen for KL or SKL cells. Referring now to FIG.2B, (right panel) analogous to murine cells, all four receptor subtypeswere expressed on the surface of human CD34⁺ UCB cells and QRT-PCRanalysis detected mRNA for all four EP receptors (FIG. 2B).

3. Short-Term PGE₂ Exposure Increases HSPC Homing Efficiency

Enhanced HSPC engraftment observed upon pulse-exposure to PGE₂ mayresult from increased HSPC number and/or cell cycle status effects onfacilitating cells or effects on HSPC homing or proliferation in thehost marrow. Irrespective of its cause a marrow niche is required forHSPC to self-renew and differentiate and it is very likely that onlyHSPC homing to these niches can provide long-term repopulation.Referring now to FIG. 3A, in order to assess HSPC homing, CFSE labeledwhole bone marrow (WBM) CD45.2 cells were pulsed with dmPGE₂ or vehiclefor 2 hours on ice, washed and injected IV in lethally irradiated CD45.2hosts. After 16 hours, total CFSE⁺ cells homing to bone marrow as wellas the number of homed events within the KL and SKL cell populationswere quantified. No difference in the percentage of CFSE⁺ cells homingto the marrow was observed between dmPGE₂ and vehicle-treated cells whentotal WBM cells were evaluated; however, significantly more SKL cellshomed to the marrow than to the control. Referring now to FIG. 3B, in acongenic model, a significantly greater percentage of SKL cells was alsoobserved for dmPGE₂-treated cells compared to vehicle-treated orun-manipulated cells. No difference in homing efficiency was seenbetween untreated and vehicle-treated cells.

Referring now to FIG. 3C, in order to determine whether the enhancingeffect of dmPGE₂ on SKL cell homing was direct or indirect, the homingof enriched HSPC in a head-to-head transplant model was compared withother cells. Highly purified SKL cells from both CD45.2 and CD45.1 micewere isolated by FACS sorting, treated with dmPGE₂ or vehicle, and 3×10⁴vehicle-treated CD45.1 cells plus 3×10⁴ dmPGE₂-treated CD45.2 cellstransplanted into CD45.1/CD45.2 mice. An additional cohort wasconcurrently transplanted with congenic strain and treatment groups wereswitched to test for any bias in strain homing. Similar to studies usingWBM, dmPGE₂ pulse-exposure of purified SKL cells increased their homingefficiency by 2-fold, strongly suggesting a direct effect of PGE₂ onHSPC. Although SKL cells are not a homogenous HSPC population, they arehighly enriched for LTRC (Okada et al, 1992; Spangrude and Scollay,1990). 4. PGE₂ increases HSPC CXCR4, and the CXCR4 antagonist AMD3100blocks enhanced homing

Referring now to FIG. 4A, the stromal-cell-derived factor-1 alpha(SDF-1a)/CXCR4 axis has been implicated in HSPC trafficking and homing.The study evaluated whether the improved homing of dmPGE₂-treated HSPCwas the result of increased SDF-1a/CXCR4 signaling. Pulse-exposure ofLin^(neg) cells to dmPGE₂ increased CXCR4 expression on KL and SKL cells(FIG. 4A); similarly, dmPGE₂ pulse exposure increased CXCR4 expressionon CD34⁺ UCB cells as expected. QRT-PCR demonstrated elevated CXCR4 mRNAlevels in dmPGE₂-treated cells compared to vehicle, with maximalelevation observed at 6 hours (data not shown).

Referring now to FIG. 4B, in order to determine if up-regulated CXCR4played a role in the enhanced homing observed after PGE₂ treatment, theselective CXCR4 antagonist AMD3100, which inhibits in vitro migration toSDF-1 and homing of HSPC in vivo was used. PGE₂ pulse-exposure increasedhoming of SKL cells by ˜2-fold, and incubation of vehicle ordmPGE₂-pulsed cells with AMD3100 reduced SKL cell homing and abrogatedthe improved homing efficiency of dmPGE₂-pulsed cells.

5. PGE₂ Decreases HSPC Apoptosis Coincident with an Increase inSurvivin.

PGE₂ treatment produced a 4-fold increase in HSPC frequency and CRU(FIG. 1), but only a 2-fold enhancement in homing (FIG. 3), suggestingthat other events are involved in enhanced engraftment. Apoptosis is animportant regulatory process in normal and malignant hematopoiesis andPGE₂ has been implicated in anti-apoptotic signalling. Moreover,activation of cAMP, a downstream signaling molecule of EP receptors,inhibits apoptosis in CD34⁺ cells. One hypothesize consistent with theseresults is that dmPGE₂ treatment affects survival and/or proliferationof HSPC, which contributes to enhanced engraftment. To evaluate aneffect of dmPGE₂ on HSPC survival, Lin^(neg) cells were pulsed with 0.1nanoMolar-1 microMolar dmPGE₂ or vehicle and cultured in serum-reducedculture medium without growth factors. Pulse-exposure to dmPGE₂ reducedapoptosis in SKL cells in a dose dependent fashion (FIG. 5A), reaching˜65% inhibition at 1 microMolar.

The inhibitor of apoptosis protein survivin is an important regulator ofapoptosis and proliferation in both normal and malignant hematopoieticcells. Referring now to FIG. 5B, these results demonstrate that PGE₂affected Survivin in HSPC. At 24 hours post-dmPGE₂-pulse, intracellularSurvivin levels were significantly higher in both murine SKL cells andCD34⁺ UCB cells (1.7 and 2.4 fold, respectively) compared to control andQRT-PCR analysis indicated elevated Survivin mRNA compared to control.

Referring now to FIG. 5C decreased active caspase-3 coincident with anincrease in Survivin was seen at 24, 48, and 72 hours post-exposure ofSKL cells to dmPGE₂ compared to control.

6. PGE₂ Treatment Increases HSPC Proliferation

Survivin regulates HSPC entry into and progression through cell cycle.Furthermore, β-catenin, implicated in HSPC proliferation andself-renewal, lies downstream of EP receptor pathways. The ability ofPGE₂ to modulate these cell cycle regulators suggests that an increasein HSPC self-renewal and proliferation might contribute to the enhancedengraftment of dmPGE₂-pulsed cells. To test this hypothesis, the cellcycle status of SKL cells pulsed with dmPGE₂ or vehicle in vitro wasanalyzed. Referring now to FIG. 6A, pulse-exposure to dmPGE₂ increasedDNA content in SKL cells, an indication of increased cell cycling (leftpanels, upper right quadrant). In 3 experiments, 60% more SKL cells werein S+G2/M phase of the cell cycle after dmPGE₂ treatment compared tocontrols (FIG. 6A right panel). No significant effect on cell cycle rateof KL or Lin^(neg) cells was seen (not shown); suggesting that dmPGE₂selectively increases the cycling state of early HSPC.

To confirm the effect of dmPGE₂ on enhancement of HSPC cell cycleobserved in vitro, marrow cells were pulsed with dmPGE₂ and injectedinto congenic mice treated with BrdU post-transplant, and the proportionof donor BrdU⁺ SKL cells was determined 16 hours later. Referring now toFIG. 6B, ˜2-fold increase in the proportion of homed SKL cells in S+G2/Mphase was observed for cells pulsed with dmPGE₂ prior to transplant,confirming that short-term exposure of HSPC to dmPGE₂ stimulates HSPC toenter and progress through cell cycle in vivo.

7. Inhibition of Endogenous PGE₂ Biosynthesis by the Dual COX1/COX2Inhibitor Indomethacin Mobilizes HSPC.

Since PGE₂ increases CXCR4 receptor expression and SDF-1/CXCR4signalling is important for trafficking and retention of HSPC in themarrow. One hypothesis consistent with these results is that inhibitionof endogenous PGE₂ biosynthesis by the dual COX1/COX2 inhibitorindomethacin would also mobilize HSPC. Referring to FIG. 7A & 7B, itshows effects of daily SC administration of 150 μg/kg indomethacin or150 ug/kg baicalein alone (FIG. 7A) or with G-CSF (FIG. 7B) for 4 dayson CFU-GM mobilization. Referring now to FIG. 7A, administration of 150μg/kg indomethacin, SC, once daily for 4 days, produced a 4-foldincrease in the number of mobilized progenitor cells. Referring now toFIG. 7B, Coadministration of indomethacin with G-CSF produced a highlysynergistic increase in peripheral blood stem cell mobilization. Thelipoxygenase inhibitor baicalein had no effect on baseline orG-CSF-induced CFU-GM mobilization, suggesting that the observed effectswere specific to inhibition of the cyclooxygenase pathway. Data areexpressed as mean±SEM CFU-GM mobilized per ml of blood for N=3 mice eachassayed individually.

8. Pulse Exposure of Murine and Human HSPC to PGE₂ Increases CXCR4Expression.

To evaluate CXCR4, Lineage^(neg) mouse bone marrow cells or CD34⁺ UCBwere treated on ice with either 1 microMolar dmPGE₂ or vehicle controlfor 2 hours, washed, and then cultured in RPMI-1640/10% HI-FBS at 37° C.for 24 hours, stained for SKL (murine cells) or CD34 (human) and CXCR4and analyzed by FACS.

Referring now to FIG. 4A, CXCR4 expression on murine KL and SKL cellsand human CD34⁺ UCB cells 24 hours after treatment with dmPGE₂. Data areexpressed as Mean±SEM % change in mean fluorescence intensity (MFI) ofCXCR4 due to treatment with dmPGE₂ or vehicle (n=3). Analysis by QRT-PCRdemonstrates a 2.65 fold increase in CXCR4 mRNA.

9. Pulse Exposure of Murine SKL Cells to PGE₂ Increases Migration toSDF-1a.

Freshly isolated Lineage^(neg) mouse bone marrow cells were pulsed withdmPGE₂ or vehicle for 2 hours, washed and resuspended in media with 10%HI-FCS and cultured at 37° C. for 16 hours. After incubation, cells werewashed, resuspended in RPMI/0.5% BSA and allowed to migrate intranswells to rmSDF-1a for 4 hours. Total cell migration was measured byflow cytometry. Referring now to FIG. 9, total SKL cell migration washigher for cells pulsed with dmPGE₂. Data are the Mean±SEM percentmigration for 3 experiments. ^(‡)P<0.05 for dmPGE₂ treated cellscompared to cells treated with vehicle.

10. Pulse Exposure of Human CD34⁺ cells to PGE₂ Increases Migration toSDF-1a.

Freshly isolated UCB CD34⁺ cells were pulsed with dmPGE₂ or vehicle for2 hours, washed and resuspended in media with 10% HI-FCS and cultured at37 ° C. for 16 hours. After incubation, cells were washed, resuspendedin RPMI/0.5% BSA and migration to rhSDF-1 measured by flow cytometry. Toblock the CXCR4 receptor, replicate cells were incubated with 5micrograms/ml AMD3100 for 30 minutes prior to the migration assay.Referring now to FIG. 10, the data are the Mean±SEM percentage migrationfor 3 experiments.

11. Blocking the CXCR4 Receptor Blocks PGE₂ Enhancement of SKL CellHoming.

To evaluate the role of CXCR4 in homing, Lineage CD45.2 cells weretreated with vehicle or 1 microMolar dmPGE₂ plus 10 microMolar AMD3100,2×10⁶ treated cells injected into lethally-irradiated CD45.1 mice andhomed SKL cells recovered 16 hours post-transplant and analyzed by FACS.Referring now to FIG. 11. homing efficiency of vehicle and dmPGE₂treated cells to bone marrow in the absence and presence of 10microMolar AMD3100. Cells were incubated with AMD3100 for 30 minutesprior to the homing assay.

12. PGE₂ Increases the Cell Cycle Rate of Murine SKL Cells In Vitro.

Lineage^(neg) cells were treated with either vehicle or 1 microMolardmPGE₂ for 2 hours, washed and cultured in media with rmSCF, rhFlt3 andrhTpo. After 20 hours cells were stained for SKL and Hoechst-33342 andPyronin-Y. The proportion of SKL cells in cell cycle were measured byFACS. Referring now to FIG. 12, representative flow plot showing cellcycle distribution of gated SKL cells and combined data for foldincrease in cell cycle for dmPGE₂-treated cells compared to vehiclecontrol from 3 experiments, Mean±SEM, n=9 mice, each assayedindividually. The proportion of SKL cells in cell cycle were measured byFACS. Representative flow plot showing cell cycle distribution of gatedSKL cells and combined data for fold increase in cell cycle fordmPGE₂-treated cells compared to vehicle control from 3 experiments,Mean±SEM, n=9 mice, each assayed individually.

13. PGE₂ Increases the Cell Cycle Rate of Highly Purified CD150⁺48′(SLAM) SKL Cells In Vitro.

Referring now to FIG. 8, table summarizing data collected usingLin^(neg) bone marrow cells treated with either 1 microMolar dmPGE₂ orvehicle for 2 hours and cultured in the presence of growth factors (50ng/ml rmSCF, 100 ng/ml each of rhFlt-3 and rhTPO) for 20 hours, werestained for SLAM SKL, Hoechst-33342 and Pyronin-Y and the proportion ofSLAM SKL cells in G_(o), G_(i), S and G₂/M phase of the cell cycledetermined by quantitation of the DNA and the RNA content by FACS. Dataare Mean±SEM for n=9 mice, each assayed individually. ^((b))Percentageof cells in G_(i)+S+G₂M; Combined data for n=9 mice. ^((*))P<0.05compared to vehicle control.

14. Pulse Exposure to PGE₂ Increases Proliferation and Cell Cycle Rateof Homed SKL Cells In Vivo.

CD45.1 Lineage^(neg) bone marrow cells were treated with dmPGE₂ orvehicle and transplanted into lethally irradiated CD45.2 mice.Immediately after transplantation, BrdU was provided in drinking waterand administered by IP injection. Bone marrow was analyzed 16 hourslater and the proportion of CD45.1⁺, SKL cells that were BrdU⁺ wasanalyzed by FACS analysis. Referring now to FIG. 13, CD45.1 Lin^(neg)bone marrow cells were treated with dmPGE₂ or vehicle and transplantedinto lethally irradiated CD45.2 mice. Immediately after transplantation,BrdU was provided in drinking water and administered by IP injection.Bone marrow was analyzed 16 hours later and the proportion of CD45.1⁺,SKL cells that were BrdU⁺ was analyzed by FACS analysis. A higherproportion of SKL cells treated with PGE₂ homed to marrow. Data areMean±SEM, n=5 per mice/group, each assayed individually.

15. Long-Term Repopulating Activity of Stem Cells is Maintained afterPGE₂ Pulse Exposure.

For head-to-head competitive analysis, WBM from CD45.1 and CD45.2 micewere treated with vehicle or dmPGE₂ and mixed with 2×10⁵ competitormarrow cells from CD45.1/CD45.2 mice at various ratios and transplantedinto lethally-irradiated CD45.1/CD45.2 mice. The proportion of CD45.1,CD45.2, and CD45.1/CD45.2 cells in PB was determined monthly. Forsecondary, tertiary and quaternary transplants, 2×10⁶ WBM frompreviously transplanted CD45.1/CD45.2 mice at a 1:1 ratio were injectedinto lethally-irradiated CD45.1/CD45.2 mice in noncompetitive fashion.The proportion of CD45.1, CD45.2, and CD45.1/CD45.2 cells in PB wasdetermined monthly. Referring now to FIG. 14, Increased chimerism ofdmPGE₂-treated cells vs. vehicle is shown for primary transplant at 20weeks (time of secondary transplant) and in a sub-cohort at 32 weeks(time of 12 week analysis of secondary transplant), for secondarytransplant at 12 weeks and 24 weeks, and likewise for tertiary andquaternary transplants, each art 12 weeks. Data for 20 week primarytransplant were from 2 pooled experiments, n=5 mice/group/experiment,each assayed individually. Data for secondary, tertiary, and quaternarytransplants were from n=5 mice/group, each assayed individually

16. Peripheral Blood Stem Cell (PBSC) Mobilization Regimens forIndomethacin and G-CSF.

Mice were given SC treatments of 150 microgram/kg indomethacin or 150microgram/kg baicalein (lipoxygenase inhibitor) in gelatin every 48hours with or without G-CSF for 4 days. CFU-GM mobilization wasdetermined as previously described (Pelus et. al., ExperimentalHematology 33 (2005) 295-307). Referring now to FIG. 7A & 7B, thecombination of the dual cyclooxygenase inhibitor Indomethacin and G-CSFsynergistically mobilize mouse HSPC. Effects of daily SC administrationof 150 μm/kg indomethacin or 150 ug/kg baicalein (lipoxygenaseinhibitor) alone (FIG. 7A) or with G-CSF (FIG. 7B) for 4 days on CFU-GMmobilization. Data are expressed as mean±SEM CFU-GM mobilized per ml ofblood for N=3 mice each assayed.

Referring now to FIG. 16, mice were given daily, bid SC injections withG-CSF (1 microgram per mouse) or G-CSF+indomethacin (50 microgram permouse) for 4 days. CFU-GM mobilization was determined as described(Pelus et. al., Experimental Hematology 33 (2005) 295-307). Mice treatedwith the combination demonstrated a larger fold increase in CFU-GM perunit of blood than animals treated with only G-CSF.

Low density mononuclear cells from the peripheral blood of micemobilized by the above regimen were analyzed for HSPC by FACS analysis.For detection of SKL and SLAM-SKL cells were stained with Sca-1-PE-Cy7,c-kit-APC, CD150-PECy5, CD48-FITC, Lineage Cocktail-Biotin, andsecondary staining with Streptavidin-APC-Cy7. Referring now to FIG. 17,analyses were performed on a BD-LSR II. Flow cytometric analysis ofphenotypically defined HSPC in peripheral blood of mice treated withG-CSF or the combination of G-CSF and Indomethacin. N=5 mice per group,each assayed individually.

17. Combination Mobilization by Indomethacin Plus AMD3100 MobilizesHSPC.

Mice were given daily, bid SC injections with vehicle or Indomethacin(50 microgram per mouse) for four days. On day 5, mice were given eithervehicle or AMD3100 (5 mg/kg). One hour later mice were sacrificed andCFU-GM mobilization was determined as previously described (Pelus et.al, Experimental Hematology 33 (2005) 295-307). Referring now to FIG.18, Mobilization of CFU-GM by vehicle or Indomethacin treatment alone(left panel). Mobilization of CFU-GM by single administration ofAMD3100, or Indomethacin treatment+AMD3100 (right panel). Data areexpressed as mean±SEM, n=5 mice per group, each assayed individually.

18. Comparison of Mobilization Efficiency Employing Indomethacin inCombination with Various Mobilization Regimens.

Mice were treated with vehicle, indomethacin (50 microgram per mouse,bid SC, 4 days), AMD3100 (5 mg/kg day 5), G-CSF (1 microgram per mouse,bid SC, 4 days), AMD3100+GROβ (5 milligram/kg and 20 milligram/kgrespectively, day 5), AMD3100+Indomethacin (Indomethacin 50 microgramper mouse, bid SC, 4 days; AMD3100 5 milligram/kg day 5), orG-CSF+Indomethacin (1 microgram and 50 microgram respectively, bid C, 4days). CFU-GM mobilization was determined as previously described (Peluset. al., Experimental Hematology 33 (2005) 295-307). Referring now toFIG. 19, CFU-GM per niL of peripheral blood plotted for varioustreatment regimes as outline in the above.

Mice were treated with vehicle, G-CSF (1 microgram per mouse, bid SC, 4days), G-CSF+Indomethacin (50 microgram per mouse, bid SC, 4 days) orG-CSF+Meloxicam (0.3 mg/kg, bid SC, 4 days). CFU-GM mobilization wasdetermined as previously described (Pelus et. al., ExperimentalHematology 33 (2005) 295-307). Referring now to FIG. 20, a bar graphillustrating a comparison of mobilization induced by Indomethacin+G-CSFand the similar acting NSAID Meloxicam+G-CSF. Data are expressed asmean±SEM, n=5 mice per group, each assayed individually.

19. Staggered Dosing with NSAID Allows for Recovery of CXCR4 Expressionon HSPC.

CD45.1 mice were mobilized with G-CSF (1 microgram per mouse, bid, SC, 4days) or G-CSF +Indomethacin (50 microgram per mouse, bid, SC, 4 days)and peripheral blood mononuclear cells (PBMC) were collected at day 5.PBMC were mixed at various ratios with CD45.2 bone marrow andtransplanted into lethally irradiated (1100 cGy, split dose) CD45.2mice. Referring now to FIG. 21, competitive repopulating units are shownat 12 weeks post transplant (left panel). Data are expressed as mean±SEM from 2 experiments, N=5 mice per group, per experiment, eachassayed individually. Since there was no improvement in engraftment withPBMC mobilized by indomethacin co-administered with G-CSF compared toG-CSF alone, it was hypothesized that deficits in homing may occur as aresult of a decrease in CXCR4 receptor expression, and that this couldbe alleviated by staggering the indomethacin and G-CSF treatments.CD45.2 mice were mobilized with G-CSF (1 microgram per mouse, bid, SC, 4days), G-CSF+Indomethacin without a stagger (50 microgram per mouse,bid, SC, 4 days), G-CSF+Indomethacin with a 1 day stagger (Indomethacinstarted first and given for 4 days, and G-CSF given for 4 days startingon the second indomethacin treatment, creating 1 day with G-CSF withoutindomethacin before collection of PBMC), or G-CSF +Indomethacin with a 2day stagger (Indomethacin started first and given for 4 days, and G-CSFgiven for 4 days starting on the third indomethacin treatment, creating2 days with G-CSF without Indomethacin before collection of PBMC).Referring now to FIG. 21 (right panel) the expression of CXCR4 on SKLcells is shown. Data are expressed as mean±SEM, N=5 mice per group, eachassayed individually.

20. Mobilized PBSC from G-CSF Plus NSAID Treated Mice Show SignificantlyEnhanced Long-Term Stem Cell Function Compared to PBSC Mobilized byG-CSF Alone.

CD45.1 mice were mobilized with G-CSF or G-CSF+Indomethacin (1 daystagger) and PBMC were transplanted with CD45.2 competitor bone marrowinto lethally irradiated CD45.2 mice. Referring now to FIG. 22,chimerism at multiple donor: competitor ratios (left panel) andcompetitive repopulating units (right panel) are shown at 12 weekspost-transplant. Data are expressed as mean±SEM, n=5 mice per group,each assayed individually.

21. PBSC from G-CSF Plus NSAID Mobilized Mice Restore Peripheral BloodNeutrophil Counts Faster when Transplanted into Lethally Irradiated MiceCompared to PBSC Mobilized by G-CSF Alone.

Mice were mobilized with G-CSF or G-CSF+Meloxicam (1 day stagger) and2×10⁶ PBMC were transplanted into lethally irradiated recipients.Neutrophils in blood were enumerated every other day by a Hemavet 950 FS(Drew Scientific) until full recovery (compared to control subset).Platelets in blood were enumerated every other day by a Hemavet 950 FS(Drew Scientific) until full recovery (compared to control subset).Referring now to FIG. 23, Neutrophils in peripheral blood (PB) wereenumerated every other day until full recovery (compared to controlsubset). These data are expressed as mean±SEM, n-10 mice per group, eachassayed individually. Referring now to FIG. 24, platelets in peripheralblood (PB) were enumerated every other day until full recovery (comparedto control subset). The data are expressed as mean±SEM, n=10 mice pergroup, each assayed individually.

22. Determining the Effect of G-CSF and Meloxicam on Cell Mobilizationin Baboons.

Referring now to FIG. 25, a first group of baboons was mobilized withthe following dosing regime treatment with 10 ug/kg of body weight ofG-CSF then after a two week wash out period the animals were treatedwith 10 ug/kg G-CSF plus 0.1 mg/kg of meloxicam. A second group ofbaboon was mobilized with the following regime 10 ug/kg G-CSF plus 0.1mg/kg of meloxicam and after a two week wash out period with 10 ug/kgG-CSF. Referring now to FIG. 26, CD34⁺ cells in PB were determined byFACS analysis and CFU-GM per ml of blood determined as previouslydescribed. Co-administering G-CSF and Meloxicam increased mobilizationof CD34⁺ cells (left panel) and CFU-GM measured per unit of blood drawn.

23. Optimal Enhancement of PBSC Mobilization in Mice Requires Inhibitionof Both COX1 and COX 2 Enzymes.

Mice were mobilized with G-CSF and CFU-GM in PB was compared tomobilization regimens with G-CSF and the combination of various NSAIDS(Aspirin [COX-1 and COX-2]; Licofelone [COX-2 and 5-LOX]; SC-560[COX-1]; Valeryl Salicylate [COX-1]; Valdecoxib [COX-2]; NS-398[COX-2]). Referring now to FIG. 27, the results of these tests aresummarized and these data are expressed as mean±SEM, n=4 mice per group,each assayed individually. Compounds that are known to inhibit both COX1and 2 were better at mobilizing colony forming cells than compounds thatare considered highly selective for only one of the two isozymes, inorder to test the efficacy of two commonly used COX inhibitors, micewere mobilized with G-CSF or the combination of G-CSF and aspirin oribuprofen (PO., bid, 4 days). CFU-GM was determined as previouslydescribed. Referring now to FIG. 28, dose-response analysis ofG-CSF+Aspirin and G-CSF+Ibuprofen mobilization of CFU-GM to peripheralblood are present in bar graph form for a control group of mice treatedwith only G-CSF; for mice treated with G-CSF plus 10, 20 or 40milligram/kg of aspirin; and for mice treated with G-CSF plus 10, 20 or40 milligram/kg of ibuprofen. Data are expressed as mean±SEM, n=4 miceper group, each assayed individually.

24. Measuring the Dose Dependent Effect of Meloxicam on CFU in Mice

Mice were mobilized with G-CSF and following doses of meloxicam 0.0(control) 0.02, 0.2, 0.5, 1.5, and 3 milligram/kg of body weight formeloxicam (bid SC, 4 days) and CFU-GM was determined as previouslydescribed. Referring now to FIG. 29 the dose response of meloxicam onHSPC was measured in samples of the animals' peripheral blood; or in theanimals' bone marrow (FIG. 30). These data are expressed as mean±SEM,n=3 mice per group, each assayed individually.

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

REFERENCES

Breyer, R. M, Bagdassarian, C. K., Myers, S. A., and Breyer, M. D.(2001). Prostanoid receptors: subtypes and signaling. Annu. Rev.Pharmacol. Toxicol. 41, 661-690.

Broxmeyer, H. E. (2006). Cord Blood Hematopoietic Stem and ProgenitorCells. In Essentials of Stem Cell Biology, Elsevier, Inc.), pp. 133-137.

Cheng, T., Rodrigues, N., Shen, H., Yang, Y., Dombkowski, D., Sykes, M,and Scadden, D. T. (2000). Hematopoietic stem cell quiescence maintainedby p21cip1/waf1. Science 287, 1804-1808.

Fleming, H. E., Janzen, V., Lo, C. C, Guo, J., Leahy, K. M., Kronenberg,H. M., and Scadden, D. T. (2008). Wnt signaling in the niche enforceshematopoietic stem cell quiescence and is necessary to preserveself-renewal in vivo. Cell Stem Cell 2, 274-283.

Fruehauf, S. and Seggewiss, R. (2003). It's moving day: factorsaffecting peripheral blood stem cell mobilization and strategies forimprovement [corrected]. Br. J. Haematol. 122, 360-375.

Fukuda, S., Mantel, C. R., and Pelus, L. M. (2004). Survivin regulateshematopoietic progenitor cell proliferation through p21 WAF1/Cip1-dependent and -independent pathways. Blood 103, 120-127.

Fukuda, S. and Pelus, L. M. (2001). Regulation of theinhibitor-of-apoptosis family member survivin in normal cord blood andbone marrow CD34(+) cells by hematopoietic growth factors: implicationof survivin expression in normal hematopoiesis. Blood 98, 2091-2100.

Goldman, J. M. and Horowitz, M. M. (2002). The international bone marrowtransplant registry. Int. J. Hematol. 76 Suppl 1, 393-397.

Hall, K. M., Horvath, T. L, Abonour, R., Cornetta, K., and Srour, E. F.(2006). Decreased homing of retro virally transduced human bone marrowCD34+cells in the NOD/SCID mouse model. Exp. Hematol. 34, 433-442.

Janzen, V., Fleming, H. E., Riedt, T., Karlsson, G., Riese, M J., Lo, C.C, Reynolds, G., Milne, C. D., Paige, C. J., Karlsson, S., Woo, M., andScadden, D. T. (2008). Hematopoietic stem cell responsiveness toexogenous signals is limited by caspase-3. Cell Stem Cell 2, 584-594.

Khan, N. I. and Bendall, L, J. (2006). Role of WNT signaling in normaland malignant hematopoiesis. Histol. Histopathol. 21, 761-774.

Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio,P. C, and Altieri, D. C. (1998). Control of apoptosis and mitoticspindle checkpoint by survivin. Nature 396, 580-584.

Liu, X. H., Kirschenbaum, A., Lu, M., Yao, S., Dosoretz, A., Holland J.F., and Levine, A. C. (2002). Prostaglandin E2 induces hypoxia-induciblefactor-1 alpha stabilization and nuclear localization in a humanprostate cancer cell line. J Biol Chem 277, 50081-50086.

Muller-Sieburg, C. E. and Sieburg, H. B. (2006). Clonal diversity of thestem cell compartment. Curr Opin Hematol 13, 243-248.

Namba, T., Sugimoto, Y., Negishi, M., Irie, A-, Ushikubi, F., Kakizuka,A-, Ito, S., Ichikawa, A., and Narumiya, S. (1993). Alternative splicingof C-terminal tail of prostaglandin E receptor subtype EP3 determinesG-protein specificity. Nature 365, 166-170.

North, T. E., Goessling, W., Walkley, C. R., Lengerke, C, Kopani. K. R.,Lord, A- M., Weber, G. J., Bowman, T. V., Jang J. H., Grosser, T.,Fitzgerald, G. A., Daley, G. Q., Orkin, S. H., and Zon, L, I. (2007).Prostaglandin E2 regulates vertebrate haematopoietic stem cellhomeostasis. Nature 447, 1007-1011.

Okada, S., Nakauchi, H., Nagayoshi, K., Nishikawa, S., Miura, Y., andSuda. T. (1992). In vivo and in vitro stem cell function of c-kit- andSea- 1 -positive murine hematopoietic cells. Blood 80, 3044-3050.

Peng, X. H., Karna, P., Cao, Z., Jiang, B. H., Zhou, M., and Yang, L.(2006). Cross-talk between epidermal growth factor receptor andhypoxia-inducible factor-1 alpha signal pathways increases resistance toapoptosis by up-regulating survivin gene expression. J Biol Chem 281,25903-25914.

Piccoli, C, D'Aprile, A., Ripoli, M., Scrima, R., Boffoli, D., Tabilio,A., and Capitanio, N. (2007). The hypoxia-inducible factor is stabilizedin circulating hematopoietic stem cells under normoxic conditions. FEBSLett 581 , 3111-3119.

Porecha, N. K., English, K., Hangoc, G., Broxmeyer, H. E., andChristopherson, K. W. (2006). Enhanced functional response to CXCL12/SDF-1 through retroviral overexpression of CXCR4 on M07e cells:implications for hematopoietic stem cell transplantation. Stem CellsDev. 75, 325-333.

Pulsipher M A, Chitphakdithai P, Logan B R, Leitman S F, Anderlini P,Klein J P, Horowitz M M, Miller J P, King R J, Confer D L, Donor,recipient, and transplant characteristics as risk factors afterunrelated donor PBSC transplantation: beneficial effects of higher CD34+cell dose. Blood. 2009 Sep. 24;114(13):2606-16. Epub 2009 Jul. 16.

Regan, J. W. (2003). EP2 and EP4 prostanoid receptor signaling. LifeSci. 74, 143-153.

Spangrude, G J. and Scollay, R. (1990). A simplified method forenrichment of mouse hematopoietic stem cells. Exp. Hematol. 75, 920-926.

Staller, P., Sulitkova, J., Lisztwan, J., Moch, H., Oakeley, E. J., andKrek, W. (2003). Chemokine receptor CXCR4 downregulated by vonHippel-Lindau tumour suppressor pVHL. Nature 425, 307-311.

Sugimoto, Y. and Narumiya, S. (2007). Prostaglandin E receptors. J.Biol. Chem. 282, 11613-11617.

Tamm, L, Wang, Y., Sausville, E., Scudiero, D. A., Vigna, N.,Oltersdorf, T., and Reed, J. C. (1998). IAP-family protein survivininhibits caspase activity and apoptosis induced by Fas (CD95), Bax,caspases, and anticancer drugs. Cancer Res. 58, 5315-5320.

Tsuboi_(j)K., SugimotOjY., and Ichikawa, A. (2002). Prostanoid receptorsubtypes. Prostaglandins Other Lipid Mediat. 68-69, 535-556.

Zagzag, D., Krishnamachary, B., Yee, H., Okuyama, H., Chiriboga, L.,Ali, M. A., Melamed J., and Semenza, G. L. (2005). Stromal cell-derivedfactor- 1 alpha and CXCR4 expression in hemangioblastoma and clearcell-renal cell carcinoma: von Hippel-Lindau loss-of-function inducesexpression of a ligand and its receptor. Cancer Res 65, 6178-6188.

1-27. (canceled)
 28. A method for gene therapy comprising: administeringto a subject in need thereof hematopoietic stem or progenitor cells thathave been: contacted ex vivo with an effective amount of a prostaglandinE2 or a derivative thereof; and transduced with a viral vector thatcontains at least one gene of interest.
 29. The method according toclaim 28, wherein the prostaglandin E2 or a derivative thereof isselected from the group consisting of PGE₂ and dmPGE₂.
 30. The methodaccording to claim 28, wherein said hematopoietic stem cells areobtained from Umbilical Cord Blood (UCB) or mobilized Peripheral Blood(PB) drawn from a donor.
 31. The method according to claim 28, whereinthe cells have been contacted with the prostaglandin E₂ or a derivativethereof for at least one hour.
 32. The method according to claim 28,wherein the cells have been contacted with the prostaglandin E₂ or aderivative thereof for about 2 hours.
 33. The method according to claim28, wherein the cells have been washed after contact with theprostaglandin E₂ or a derivative thereof
 34. The method according toclaim 28, wherein the cells have been washed prior to being administeredto the subject.
 35. A human hematopoietic stem or progenitor cell thathas been contacted ex vivo with an effective amount of a prostaglandinE₂ or a derivative thereof and that comprises a viral vector thatcontains at least one gene of interest.
 36. The human hematopoietic stemor progenitor cell according to claim 35, wherein the prostaglandin E₂or a derivative thereof is selected from the group consisting of PGE₂and dmPGE₂.
 37. The human hematopoietic stem or progenitor cellaccording to claim 35, wherein the cell has been contacted with theprostaglandin E₂ or a derivative thereof for about one to about 6 hours.38. The human hematopoietic stem or progenitor cell according to claim35, wherein the cell has been contacted with the prostaglandin E₂ or aderivative thereof for about 2 hours.
 39. The human hematopoietic stemor progenitor cell according to claim 35, wherein the cell has beenwashed after contact with the prostaglandin E₂ or a derivative thereof.40. The human hematopoietic stem or progenitor cell according to claim35, wherein the cell has been washed prior to being transplanted into asubject.
 41. The human hematopoietic stem or progenitor cell accordingto claim 35, wherein the human hematopoietic stem cell is CD34+.
 42. Acell transplant for gene therapy comprising: hematopoietic stem and/orprogenitor cells contacted ex vivo with an effective amount of aprostaglandin E₂ or a derivative thereof and transduced with a viralvector that contains at least one gene of interest.
 43. The celltransplant according to claim 42, wherein the cells are contacted withthe prostaglandin E₂ or a derivative thereof for at least one hour. 44.The cell transplant according to claim 42, wherein the cells arecontacted with the prostaglandin E₂ or a derivative thereof for aboutone to about 6 hours.
 45. The cell transplant according to claim 42,wherein the prostaglandin E₂ or a derivative thereof is selected fromthe group consisting of PGE₂ and dmPGE₂.
 46. The cell transplantaccording to claim 42, wherein the human hematopoietic stem cells areLineage negative (Lin^(Neg)) cells.
 47. The cell transplant according toclaim 42, wherein the human hematopoietic stem cells are CD34+ cells.